Bottom Line:
Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers.We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates.These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.

ABSTRACTAlzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

Figure 1: Kinetics of Aβ42 aggregation in the presence of Brichos(a-c) Reaction profiles from left (blue) to right (green) for aggregation in the absence of Brichos and in the presence of 10%, 15%, 35%, 50%, 75%, and 100% Aβ42 monomer equivalents of Brichos. The data show averages (points) and standard errors over five technical replicas. The effect of Brichos saturates at a stoichiometry of approximately one monomer equivalent of Aβ42 (see Supplementary Fig.1). The blue dashed line is the integrated rate law for Aβ42 aggregation in the absence of Brichos using the rate constants determined previously19. The green dashed lines show predictions for the resulting reaction profiles when each of (a) primary nucleation, (b) fibril elongation, and (c) secondary nucleation are inhibited by the chaperone (see Supplementary Fig. 2). The thin dotted lines in (c) are theoretical predictions for the reaction profiles at the intermediate Brichos concentrations using the association and dissociation rate constants determined for its binding by means of SPR (Fig. 3b). (d-f) Time evolution of the nucleation rate calculated from the kinetic analysis. The blue line corresponds to the situation in the absence of Brichos and the green dashed lines show predictions for the cases when each of (d) primary nucleation, (e) fibril elongation, and (f) secondary nucleation are inhibited by the chaperone. The insets show the relative number of oligomers generated during the aggregation reaction. The concentration of monomeric Aβ42 was 3μM.

Mentions:
We first monitored the kinetics of the aggregation of Aβ42 under the quiescent conditions described previously19,20, and obtained data completely consistent with this earlier study. In order to establish well-defined initial conditions for the kinetic experiments, and hence to obtain reproducible data, we purified to very high levels the recombinant monomeric Aβ42 peptide by repeated applications of size-exclusion chromatography and controlled carefully the inertness of surfaces with which it made contact20. On addition of the Brichos domain from proSP-C50, at concentrations between 10% and 100% Aβ42 monomer equivalents, we observed a reduction in the overall rate of formation of fibrils and characteristic changes in the time course of the reaction (Fig. 1; see also Supplementary Fig. 1). To connect these observations with the underlying microscopic mechanisms that contribute to the macroscopic changes, we used an analytical approach to predict the effects of perturbing the different individual microscopic processes involved in Aβ42 aggregation on the global kinetics of aggregation51. Specifically, alterations in the rates of primary nucleation, fibril elongation, and secondary nucleation are each predicted to result in distinct and characteristic changes in the shape of the reaction profiles (Fig. 1).

Figure 1: Kinetics of Aβ42 aggregation in the presence of Brichos(a-c) Reaction profiles from left (blue) to right (green) for aggregation in the absence of Brichos and in the presence of 10%, 15%, 35%, 50%, 75%, and 100% Aβ42 monomer equivalents of Brichos. The data show averages (points) and standard errors over five technical replicas. The effect of Brichos saturates at a stoichiometry of approximately one monomer equivalent of Aβ42 (see Supplementary Fig.1). The blue dashed line is the integrated rate law for Aβ42 aggregation in the absence of Brichos using the rate constants determined previously19. The green dashed lines show predictions for the resulting reaction profiles when each of (a) primary nucleation, (b) fibril elongation, and (c) secondary nucleation are inhibited by the chaperone (see Supplementary Fig. 2). The thin dotted lines in (c) are theoretical predictions for the reaction profiles at the intermediate Brichos concentrations using the association and dissociation rate constants determined for its binding by means of SPR (Fig. 3b). (d-f) Time evolution of the nucleation rate calculated from the kinetic analysis. The blue line corresponds to the situation in the absence of Brichos and the green dashed lines show predictions for the cases when each of (d) primary nucleation, (e) fibril elongation, and (f) secondary nucleation are inhibited by the chaperone. The insets show the relative number of oligomers generated during the aggregation reaction. The concentration of monomeric Aβ42 was 3μM.

Mentions:
We first monitored the kinetics of the aggregation of Aβ42 under the quiescent conditions described previously19,20, and obtained data completely consistent with this earlier study. In order to establish well-defined initial conditions for the kinetic experiments, and hence to obtain reproducible data, we purified to very high levels the recombinant monomeric Aβ42 peptide by repeated applications of size-exclusion chromatography and controlled carefully the inertness of surfaces with which it made contact20. On addition of the Brichos domain from proSP-C50, at concentrations between 10% and 100% Aβ42 monomer equivalents, we observed a reduction in the overall rate of formation of fibrils and characteristic changes in the time course of the reaction (Fig. 1; see also Supplementary Fig. 1). To connect these observations with the underlying microscopic mechanisms that contribute to the macroscopic changes, we used an analytical approach to predict the effects of perturbing the different individual microscopic processes involved in Aβ42 aggregation on the global kinetics of aggregation51. Specifically, alterations in the rates of primary nucleation, fibril elongation, and secondary nucleation are each predicted to result in distinct and characteristic changes in the shape of the reaction profiles (Fig. 1).

Bottom Line:
Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers.We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates.These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

Affiliation:
Department of Chemistry, University of Cambridge, Cambridge, UK.

ABSTRACTAlzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.